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Comparative Genomics of Rickettsia prowazekii Madrid E and Breinl Strains. Hong Ge, 2004.Rickettsia prowazekii, the causative agent of epidemic typhus, has been responsible for millions of human deaths . Madrid E is an attenuated strain of R . prowazekii, while Breinl is a virulent strain . The genomic DNA sequence of Madrid E has recently been published . To study the genomic variations between Madrid E (reference) and Breinl (test) DNAs, cohybridization experiments were performed on a DNA microarray containing all 834 protein-coding genes of Madrid E . Of the 834 genes assessed, 24 genes showed 1.5- to 2.0-fold increases in hybridization signals in Breinl DNA compared to Madrid E DNA, indicating the presence of genomic variations in  3%
of the total genes . Eighteen of these 24 genes are predicted to be
involved in different functions . Southern blot analysis of five
genes, virB4, ftsK, rfbE, lpxA, and rpoH,
suggested the presence of an additional paralog(s) in Breinl,
which might be related to the observed increase in hybridization
signals . Studies by real-time reverse transcription-PCR revealed an
increase in expression of the above-mentioned five genes and five
other genes . In addition to the elevated hybridization signals of 24
genes observed in the Breinl strain, one gene (rp084) showed
only 1/10 the hybridization signal of Madrid E . Further analysis of
this gene by PCR and sequencing revealed a large deletion flanking
the whole rp084 gene and part of the rp083 gene in the
virulent Breinl strain . The results of this first rickettsial DNA
microarray may provide some important information for the elucidation
of pathogenic mechanisms of R . prowazekii .
Dissimilatory Arsenate Reduction with Sulfide as Electron Donor: Experiments with Mono Lake Water and Isolation of Strain MLMS-1, a Chemoautotrophic Arsenate Respirer. Shelley E. Hoeft, 2004.Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors . Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction . We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water . Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide . No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide . The rate of arsenate reduction in lake water was dependent on the amount of available arsenate . We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution . The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite . Chemoautotrophy was confirmed by the incorporation of H14CO3– into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products . Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the  -Proteobacteria, located near sulfate reducers like Desulfobulbus sp . (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake . However, strain MLMS-1 does not grow with sulfate as its electron acceptor .
Metabolic Primers for Detection of (Per)chlorate-Reducing Bacteria in the Environment and Phylogenetic Analysis of cld Gene Sequences. Kelly S. Bender, 2004.Natural attenuation of the environmental contaminant perchlorate is a cost-effective alternative to current removal methods . The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within a target site . To detect DPRB in the environment, two degenerate primer sets targeting the chlorite dismutase (cld) gene were developed and optimized . A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method . Screening of environmental samples indicated that all products amplified by this method were cld gene sequences . These sequences were obtained from pristine sites as well as contaminated sites from which DPRB were isolated . More than one cld phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites . The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation . Sequences of cld genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene . Comparisons of the cld and 16S ribosomal DNA (rDNA) gene trees indicated that the cld gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration . Regulatory Response of Methanococcus maripaludis to Alanine, an Intermediate Nitrogen Source. Thomas J. Lie, 2002.In the methanogenic archaeon Methanococcus maripaludis, growth with ammonia results in conditions of nitrogen excess . Complete repression of nitrogen fixation (nif) gene transcription occurs, and glutamine synthetase (glnA) gene transcription falls to a basal constitutive level . In addition, ammonia completely switches off nitrogenase enzyme activity . In contrast, growth with dinitrogen as the sole nitrogen source results in nitrogen starvation, full expression of nif and glnA, and high activity of nitrogenase . Here we report that a third nitrogen source, alanine, results in an intermediate regulatory response . Growth with alanine resulted in intermediate transcription of nif and glnA, and addition of alanine to a nitrogen-fixing (diazotrophic) culture caused partial switch-off of nitrogenase . This uniformity of response occurred despite differences in regulatory mechanisms . Nitrogenase switch-off requires the nitrogen sensor homologs NifI1 and NifI2, while transcriptional regulation of nif and glnA relies on a different, unknown sensor mechanism . In addition, although nif and glnA transcription are governed by a common repressor, the numbers and arrangements of repressor binding sites differ . Thus, the nif promoter region contains two operators situated downstream of the transcription start site, while the glnA promoter region contains only one operator just upstream of two closely spaced transcription start sites . In a previous study of nif expression using ammonia, we were able to detect a role only for the first nif operator in repression . Here we show that nif repression by alanine requires the second operator as well . In contrast, in the case of glnA the single operator was sufficient for repression by ammonia or alanine . These results suggest a uniform cellular response to nitrogen that is mediated by a different mechanism in each case . Strictly Polyphosphate-Dependent Glucokinase in a Polyphosphate-Accumulating Bacterium, Microlunatus phosphovorus. Shotaro Tanaka, 2003.ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK) . Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus . The polyP-GK produced glucose-6-Pi from glucose and polyP, but it could not phosphorylate glucose with ATP . The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis . Effects of Light on the Microcystin Content of Microcystis Strain PCC 7806. Claudia Wiedner, 2003.Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans . The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures . Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 µmol of photons m-2 s-1 on a light-dark rhythm of 12 h -12 h . The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale . The cellular microcystin content varied between 34.5 and 81.4 fg cell-1 and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth . Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth . The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR . Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days . During the period of illumination at a PAR of 10 and 40 µmol of photons m-2 s-1, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period . Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 µmol of photons m-2 s-1 than at 10 µmol of photons m-2 s-1 and did not change significantly during the light-dark cycles at both irradiances . In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited . Infection of Acanthamoeba castellanii with Mycobacterium bovis and M . bovis BCG and Survival of M . bovis within the Amoebae. Stephanie J. Taylor, 2003.Survival of Mycobacterium bovis after ingestion by protozoa would provide an environmental reservoir for infection of cattle . We have shown that M . bovis survived ingestion by Acanthamoeba castellanii . In contrast, two strains of M . bovis BCG did not survive well within Acanthamoeba.
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